Hardy Katharine M, Hoffman Emely A, Gonzalez Pedro, McKay Brian S, Stamer W Daniel
Department of Cell Biology, the University of Arizona, Tucson, AZ 85724, USA.
J Biol Chem. 2005 Aug 12;280(32):28917-26. doi: 10.1074/jbc.M504803200. Epub 2005 Jun 7.
Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.
肌纤蛋白（MYOC）是一种具有广泛表达模式但功能未知的蛋白质。MYOC与与分泌小泡一致的细胞内结构相关联，然而，在大多数研究的细胞类型中，MYOC局限于细胞内区室。在小梁网中，MYOC与细胞内小泡相关联，但也存在于细胞外空间。本研究的目的是更好地理解小梁网细胞中MYOC细胞外转运的机制。使用生化方法，我们发现MYOC在细胞内定位于胞质和颗粒部分。当在线性蔗糖梯度上分离细胞内膜时，MYOC在比传统分泌小泡和溶酶体密度更低的部分达到平衡。在随时间追踪新生MYOC的脉冲标记实验中，即使在布雷菲德菌素A存在的情况下，通过SDS-PAGE观察到的MYOC特征性双峰也没有改变；这表明MYOC没有糖基化，也不是通过传统分泌机制释放的。当检测来自人小梁网细胞的条件培养基时，天然和重组的MYOC都与具有外泌体生化特征且含有主要组织相容性复合体II类抗原HLA-DR的细胞外膜群体相关联。MYOC与类似外泌体膜的关联似乎是特异性的，位于细胞外表面，并且是可逆的。综上所述，数据表明MYOC通过一种非传统机制出现在小梁网细胞的细胞外空间，可能与类似外泌体的小泡有关。
